Purine nucleotides have a multi-functional role in cellular metabolism as suggested by the following selected list: they are directly involved in most kinase reactions, they function as regulators of many allosteric enzymes, they have been implicated (in the form of cyclic AMP) in the action of several hormones, and they participate in dehydrogenase reactions as part of the DPN and TPN molecules. The broad objective of this study is to develop the tools which will allow a systematic exploration of the amino acid residues in the coenzyme binding sites and purine nucleotide-specific regulatory sites of dehydrogenases. Using the general approach of affinity labeling, new reagents will be synthesized which incorporate, at known positions of the purine or ribose ring, alkylating agents capable of reacting covalently with the protein; the purine nucleotide or pyridine nucleotide moiety will thus provide the specificity for chemical modification of the proteins within the binding sites. It is proposed to explore at least three enzymes by means of these reagents: bovine glutamate dehydrogenase and the DPN and TPN-specific isocitrate dehydrogenases from mammalian heart muscle. The catalytic activity of glutamate dehydrogenase is observed with either DPN or TPN, and is regulated by several purine nucleotides, as typified by ADP activation and GTP inhibition. The DPN-dependent isocitrate dehydrogenase is also an allosteric enzyme activated by ADP; whereas the TPN-specific enzyme is not known to be subject to control by nucleotide modifiers. A detailed investigation will be conducted for each enzyme of the nature and reactivity of the functional groups in the coenzyme and allosteric sites in order to ascertain a chemical basis for the purine nucleotide specificity. This same approach may be extended to other dehydrogenases as well as to other classes of protein which interact with purine nucleotides.